Gap function communication is a function that enables neighboring cells to directly exchange small cytosolic molecules. This function is mediated by channels that span two plasma membranes and a narrow extracellular space (gap). A cell-cell channel is made of two hemichannels (connexons) composed of six connexin (Cx) proteins. Gap junction communication is an essential mean of functional integration in cell communities. Indeed, its malfunction has serious consequences to organ function, such as cardiac arrhythmia, uterine dysfunction, X- linked Charcot-Marie-Tooth demyelinating disease (CMTX1), cardiac malformation, congenital deafness, and so on. Over the years, much has been learned on gap junction structure and function, but crucial aspects of channel regulation (gating) are still unclear. This proposal is aimed at improving our understanding of the molecular basis of channel gating through a multiple approach that includes: fluorescence imaging, two-hybrid assay, Freeze- fracture Replica Immunolabeling (FRIL), immunoprecipitation, Cx and calmodulin (CaM) mutagenesis, channel expression in Xenopus oocyte and cultured HeLa cells, and electrophysiological recording of junctional conductance and single-channel activity. Much of the proposal is based on our recent data demonstrating that CaM is associated with gap junctions and plays a direct role in chemical gating of Cx32 channels, possibly by physically obstructing the pore (cork model). The interaction between Cx32 and CaM will be defined by expressing Cx32 and CaM linked to fluorescent proteins and by testing for colocalization and fluorescence-resonance-energy-transfer (FRET, positive for fluorophores less than 100 delta apart). Cx-CaM association will also be probed by FRIL, immunofluorescence and co- immunoprecipitation. The single-channel behavior of Cx32 mutants expressed in the presence and absence of CaM mutants will be monitored in HeLa cells. The role of CaM components in gating will be defined by expressing CaM mutants varying in central helix length and N-terminus deleted CaM. The relevance of a potential CaM binding site identified in connexins (similar to that of CaM kinase II) will be tested with deletion and mutation of crucial Cx32 residues.